1. Field of the Invention
The present invention concerns a novel avian hepatitis E virus, immunogenic compositions, diagnostic reagents, vaccines and methods of detecting or protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis E.
2. Description of the Related Art
Human hepatitis E is an important public health disease in many developing countries, and is also endemic in some industrialized countries. Hepatitis E virus (hereinafter referred to as “HEV”), the causative agent of human hepatitis E, is a single positive-stranded RNA virus without an envelope (R. H. Purcell, “Hepatitis E virus,” FIELDS VIROLOGY, Vol. 2, pp. 2831-2843, B. N. Fields et al. eds, Lippincott-Raven Publishers, Philadelphia (3d ed. 1996)). The main route of transmission is fecal-oral, and the disease reportedly has a high mortality rate, up to 20%, in infected pregnant women. The existence of a population of individuals who are positive for HEV antibodies (anti-HEV) in industrialized countries and the recent identification of numerous genetically distinct strains of HEV have led to a hypothesis that an animal reservoir for HEV exists (X. J. Meng, “Zoonotic and xenozoonotic risks of hepatitis E virus,” Infect. Dis. Rev. 2:35-41 (2000); X. J. Meng, “Novel strains of hepatitis E virus identified from humans and other animal species: Is hepatitis E a zoonosis?” J. Hepatol. 33:842-845 (2000)). In 1997, the first animal strain of HEV, swine hepatitis E virus (hereinafter referred to as “swine HEV”), was identified and characterized from a pig in the U.S. (X. J. Meng et al., “A novel virus in swine is closely related to the human hepatitis E virus,” Proc. Natl. Acad. Sci. USA 94:9860-9865 (1997)). Swine HEV was shown to be very closely related genetically to human HEV. Interspecies transmission of HEV has been documented: swine HEV infects non-human primates and a U.S. strain of human HEV infects pigs. These data lend further credence to the hypothesis of an animal reservoir for HEV.
Numerous genetically distinct strains of HEV have been identified from patients with acute hepatitis in both developing and industrialized countries. The two U.S. strains of human HEV recently identified from hepatitis E patients (US-1 and US-2) are genetically distinct from other known HEV strains worldwide but are closely related to each other and to the U.S. strain of swine HEV (J. C. Erker et al., “A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques,” J. Gen. Virol. 80:681-690 (1999); X. J. Meng et al., “Genetic and experimental evidence for cross-species infection by the swine hepatitis E virus,” J. Virol. 72:9714-9721 (1998); G. G. Schlauder et al., “The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States,” J. Gen. Virol. 79:447-456 (1998)). Similarly, several isolates of HEV have been identified from patients in Taiwan with no history of travel to endemic region. An Italian strain of human HEV was found to share only about 79.5 to 85.8% nucleotide sequence identity with other known strains of HEV. Schlauder et al. recently identified another Italian and two Greek strains of HEV (G. G. Schlauder et al., “Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV,” J. Med. Virol. 57:243-51 (1999)). The sequences of the Greek and Italian strains of HEV differed significantly from other known strains of HEV. In endemic regions, strains of HEV, which are distinct from the previously known epidemic strains, have also been identified in Pakistan (H. Van Cuyck-Gandre et al., “Short report: phylogenetically distinct hepatitis E viruses in Pakistan,” Am. J. Trop. Med. Hyg. 62:187-189 (2000)), Nigeria (Y. Buisson et al., “Identification of a novel hepatitis E virus in Nigeria,” J. Gen. Virol. 81:903-909 (2000)) and China (Y. Wang et al., “A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis,” J. Gen. Virol. 80:169-77 (1999); Y. Wang et al., “The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3,” J. Gen. Virol. 81:1675-1686 (2000)). Six isolates of HEV were identified from Chinese hepatitis E patients that were negative for anti-HEV assayed by the serological test used (Y. Wang et al., 1999, supra). The intriguing fact is that these recently identified strains of HEV are genetically distinct from each other and from other known strains of HEV. Although the source of these human HEV strains is not clear, it is plausible that they may be of animal origins.
Recently, several U.S. patents have issued which concern the human hepatitis E virus. U.S. Pat. No. 6,022,685 describes methods and compositions for detecting anti-hepatitis E virus activity via antigenic peptides and polypeptides. U.S. Pat. No. 5,885,768 discloses immunogenic peptides which are derived from the ORF1, ORF2 and ORF3 regions of hepatitis E virus, diagnostic reagents containing the peptide antigens, vaccines and immunoreactive antibodies. U.S. Pat. No. 5,770,689 relates to certain ORF Z peptides of the human HEV genome. U.S. Pat. No. 5,741,490 deals with a vaccine and vaccination method for preventing hepatitis E viral infections. U.S. Pat. No. 5,686,239 provides a method of detecting HEV antibodies in an individual using a peptide antigen obtained from the human HEV sequence.
Evidence of HEV infection of domestic and farm animals has been well documented (X. J. Meng, “Zoonotic and xenozoonotic risks of hepatitis E virus,” Infect. Dis. Rev. 2:35-41 (2000); X. J. Meng, “Novel strains of hepatitis E virus identified from humans and other animal species: Is hepatitis E a zoonosis?” J. Hepatol., 33:842-845 (2000); R. H. Purcell, “Hepatitis E virus,” FIELDS VIROLOGY, Vol. 2, pp. 2831-2843, B. N. Fields et al. eds, Lippincott-Raven Publishers, Philadelphia (3d ed. 1996)). Anti-HEV was detected in pigs from developing countries such as Nepal (E. T. Clayson et al., “Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal,” Am. J. Trop. Med. Hyg. 53:228-232 (1995)), China (X. J. Meng et al., “Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population,” J. Med. Virol. 58:297-302 (1999)) and Thailand (id), and from industrialized countries such as U.S. (X. J. Meng et al., “A novel virus in swine is closely related to the human hepatitis E virus,” Proc. Natl. Acad. Sci. USA 94:9860-9865 (1997)), Canada (X. J. Meng et al., 1999, supra), Korea (X. J. Meng et al., 1999, id), Taiwan (S. Y. Hsieh et al., “Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus,” J. Clin. Microbiol. 37:3828-3834 (1999)), Spain (S. Pina et al., “HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain,” J. Hepatol. 33:826-833 (2000)) and Australia (J. D. Chandler et al., “Serological evidence for swine hepatitis E virus infection in Australian pig herds,” Vet. Microbiol. 68:95-105 (1999)). In addition to pigs, Kabrane-Lazizi et al. reported that about 77% of the rats from Maryland, 90% from Hawaii and 44% from Louisiana are positive for anti-HEV (Y. Kabrane-Lazizi et al., “Evidence for wide-spread infection of wild rats with hepatitis E virus in the United States,” Am. J. Trop. Med. Hyg. 61:331-335 (1999)). Favorov et al. also reported the detection of IgG anti-HEV among rodents in the U.S. (M. O. Favorov et al., “Prevalence of antibody to hepatitis E virus among rodents in the United States,” J. Infect. Dis. 181:449-455 (2000)). In Vietnam where HEV is endemic, anti-HEV was reportedly detected in 44% of chickens, 36% of pigs, 27% of dogs and 9% of rats (N. T. Tien et al., “Detection of immunoglobulin G to the hepatitis E virus among several animal species in Vietnam,” Am. J. Trop. Med. Hyg. 57:211 (1997)). About 29 to 62% of cows from Somali, Tajikistan and Turkmenistan (HEV endemic regions), and about 42 to 67% of the sheep and goats from Turkmenistan and 12% of cows from Ukraine (a non-endemic region) are positive for anti-HEV (M. O. Favorov et al., “Is hepatitis E an emerging zoonotic disease?” Am. J. Trop. Med. Hyg. 59:242 (1998)). Naturally acquired anti-HEV has also been reported in rhesus monkeys (S. A. Tsarev et al, “Experimental hepatitis E in pregnant rhesus monkeys: failure to transmit hepatitis E virus (HEV) to offspring and evidence of naturally acquired antibodies to HEV,” J. Infect. Dis. 172:31-37 (1995)). These serological data strongly suggest that these animal species are infected with HEV or a related agent. Until recently, the source of seropositivity in these animals could not be definitively demonstrated since the virus was either not recovered from these animal species or the recovered virus was not genetically characterized to confirm its identity. The first and only animal strain of HEV that has been identified and extensively characterized thus far is swine HEV (X. J. Meng et al., “A novel virus in swine is closely related to the human hepatitis E virus,” Proc. Natl. Acad. Sci. USA 94:9860-9865 (1997); X. J. Meng et al., “Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV,” Arch. Virol. 143:1405-1415 (1998); X. J. Meng et al., “Genetic and experimental evidence for cross-species infection by the swine hepatitis E virus,” J. Virol. 72:9714-9721 (1998); X. J. Meng et al., “Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population,” J. Med. Virol. 58:297-302 (1999)). However, because swine HEV causes only subclinical infection and mild microscopic liver lesions in pigs, it does not provide a good, adaptable animal model to study human HEV replication and pathogenesis.
Since the identification and characterization of the first animal strain of HEV (swine HEV) in the U.S. in 1997, several other HEV strains of animal origins were genetically identified. Hsieh et al. identified a second strain of swine HEV from a pig in Taiwan (S. Y. Hsieh et al., “Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus,” J. Clin. Microbiol. 37:3828-3834 (1999)). This Taiwanese strain of swine HEV shared 97.3% nucleotide sequence identity with a human strain of HEV identified from a retired Taiwanese farmer but is genetically distinct from other known strains of HEV including the U.S. strain of swine HEV. Recently, Pina et al. identified a strain of HEV (E11 strain) from sewage samples of animal origin from a slaughterhouse that primarily processed pigs in Spain (S. Pina et al., “HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain,” J. Hepatol. 33:826-833 (2000)). The E11 strain of possible animal origin is most closely related to two Spanish strains of human HEV, and is more closely related to the U.S. swine and human strains compared to other HEV strains worldwide (id.). In addition to pigs, a strain of HEV was reportedly identified from tissue and fecal samples of wild-trapped rodents from Kathmandu Valley, Nepal (S. A. Tsarev et al., “Naturally acquired hepatitis E virus (HEV) infection in Nepalese rodents,” Am. J. Trop. Med. Hyg. 59:242 (1998)). Sequence analyses revealed that the HEV sequence recovered from Nepalese rodents is most closely related to the HEV isolates from patients in Nepal (id.).
Hepatitis-splenomegaly syndrome (hereinafter referred to as “HS syndrome”) is an emerging disease in chickens in North America. HS syndrome in chickens was first described in 1991 in western Canada, and the disease has since been recognized in eastern Canada and the U.S. HS syndrome is characterized by increased mortality in broiler breeder hens and laying hens of 30-72 weeks of age. The highest incidence usually occurs in birds between 40 to 50 weeks of age, and the weekly mortality rate can exceed 1%. Prior to sudden death, diseased chickens usually are clinically normal, with pale combs and wattles although some birds are in poor condition. In some outbreaks, up to 20% drop in egg production was observed. Affected chickens usually show regressive ovaries, red fluid in the abdomen, and enlarged liver and spleen. The enlarged livers are mottled and stippled with red, yellow and tan foci. Similar to the microscopic lesions found in the livers of humans infected with HEV, microscopic lesions in the livers of chickens with HS syndrome vary from multifocal to extensive hepatic necrosis and hemorrhage, with infiltration of mononuclear cells around portal triads. Microscopic lesions in the spleen include lymphoid depletion and accumulation of eosinophilic materials. Numerous other names have been used to describe the disease such as necrotic hemorrhage hepatitis-splenomegaly syndrome, chronic fulminating cholangiohepatitis, necrotic hemorrhagic hepatomegalic hepatitis and hepatitis-liver hemorrhage syndrome.
The cause of HS syndrome is not known. A viral etiology for HS syndrome has been suspected but attempts to propagate the virus in cell culture or embryonated eggs were unsuccessful (J. S. Jeffrey et al., “Investigation of hemorrhagic hepatosplenomegaly syndrome in broiler breeder hens,” Proc. Western Poult. Dis. Conf., p. 46-48, Sacramento, Calif. (1998); H. L. Shivaprasad et al., “Necrohemorrhagic hepatitis in broiler breeders,” Proc. Western Poult. Dis. Conf, p. 6, Sacramento, Calif. (1995)). The pathological lesions of HS in chickens, characterized by hepatic necrosis and hemorrhage, are somewhat similar to those observed in humans infected with HEV (R. H. Purcell, “Hepatitis E virus,” FIELDS VIROLOGY, Vol. 2, pp. 2831-2843, B. N. Fields et al. eds, Lippincott-Raven Publishers, Philadelphia (3d ed. 1996); C. Riddell, “Hepatitis-splenomegaly syndrome,” DISEASE OF POULTRY, p. 1041 (1997)). Since anti-HEV was detected in 44% of chickens in Vietnam (N. T. Tien et al., “Detection of immunoglobulin G to the hepatitis E virus among several animal species in Vietnam,” Am. J. Trop. Med. Hyg. 57:211 (1997)), suggesting that chickens have been infected by HEV (or a related agent), it would be advantageous to find a link between HEV infection and HS syndrome in chickens. The link would permit the development of diagnostic assays and vaccines to protect against both human and chicken HEV infections thereby providing substantial public health and veterinary benefits. These goals and other desirable objectives are met by the isolation, genetic identification and characterization of the novel avian hepatitis E virus as described herein.